Dendritic cells (DCs) are heterogeneous cells that act as the sentinels of the immune system. They recognise pathogens and their products via the expression of numerous cell surface, endosomal and cytoplasmic pattern recognition receptors (PRR) that are differentially expressed amongst different DC subsets (Diebold 2009; Hochrein and O'Keeffe 2008; Luber et al. 2010). The differential expression of PRR lends different DC subsets with the ability to recognise pathogens via distinct and overlapping mechanisms.
DCs can be divided into two major categories: conventional DCs (cDC) and plasmacytoid DCs (pDCs). The term “classic” or “conventional” DCs (cDCs) has recently been used to oppose lymphoid organ-resident DCs to pDCs. Non-lymphoid organ DCs, on the other hand are mainly called tissue DCs. While non-lymphoid tissue DCs are also different from pDCs, and primary non-lymphoid tissue DCs can be found in lymph nodes on migration but are not cDCs, the tem cDCs refers to all non-pDCs whether they are present in lymphoid or non-lymphoid tissues.
Mouse cDC can be recognized by the expression of high levels of CD11c and MHCII (Hochrein and O'Keeffe 2008). Mouse pDC have a characteristic high production of type I Interferon and have the phenotype CD11cintMHCIIloCD11b−CD205−.
Mouse and human pDC share many surface molecules, such as CD45RA, CD45RB, and CD68. They also express a similar pattern of intracellular Toll-like receptors. Expression of CD11b is able to differentiate two different subsets of cDC in human.
There is a close relationship between human and mouse DC (O'Keeffe et al., 2003). Mouse blood cells with the phenotype CD11cloCD11b−CD45RAhi closely resemble human pDC by morphology and function. In human blood, CD11c−IL3R+CD45RA+ pDC can be found, which can produce a large amount of interferon upon stimulation.
The cDC can be further divided into several subsets based upon tissue location and surface phenotype. In mice, the surface markers CD4 and CD8+ are useful markers to distinguish functionally different DC subsets. The unifying function of all cDC is the ability to induce naive T cells into the cell cycle. The exceptional ability of cDC to process and present antigen in the context of MHCI and MHCII endows them with the title ‘professional antigen presenting cells’. The CD8α+ cDC have the additional feature of ‘cross-presentation’, the ability to present exogenous antigen in the context of MHCI (Villadangos and Schnorrer 2007). The functions of cDC also extend to cytokine and chemokine production. High IL-12p70 production is a hallmark of CD8α+ cDC and high levels of chemokines including RANTES, MIP-1α and MIP-1β are produced by CD8α− cDC (Hochrein et al. 2001; Maldonado-Lopez et al. 1999; Proietto et al. 2004). Additionally IL-6, IL-8, IL-10, IL-15, IL-18, IL-23, IL-27 and TNF-α have been reported to be expressed by cDC under different stimulatory conditions.
On the other hand the pDC, generally considered as part of the DC ‘family’, lack typical cDC characteristics, including surface phenotype and morphology and also normally lack the ability to stimulate naive T cells (O'Keeffe et al. 2002). If given specific PRR stimuli they can induce some T cell division, more than B cells or macrophages, but typically in the order of 10-fold or less than that of the cDC (Villadangos and Young 2008). Unlike cDC the pDC continually present antigens on MHCII molecules once they are activated and, as a result, can continue to present new viral antigens during the course of infection (Young et al. 2008). The importance of this function of pDC during an ongoing infection is not yet elucidated. Instead the pDC, also referred to as natural interferon-producing cells (NIPC), are renowned for their production of Type I interferons (IFN-I) in response to viral or bacterial stimuli and mimics thereof (Gilliet et al. 2008; Kadowaki 2009). The categorization of the pDC as a member of the DC ‘family’ rests upon morphological and phenotypical features that they display upon activation. Namely, the pDC upregulate co-stimulation markers and MHC molecules to levels resembling the cDC and they rapidly acquire the typical stellate morphology of cDC. Based on these features, it was initially proposed that this IFN-I producing DC subset would be the ultimate anti-viral cell, combining within the same cell the innate IFN response and potent CTL stimulation. To date, these high hopes have not been realised. The IFN-I response of pDC is remarkable, but their concomitant ability to induce CTL is in most cases relatively poor.
The past 10 years have seen an explosion in the knowledge of innate recognition of pathogens. The discovery of an increasing number of PRR, including the Toll-like receptor (TLR) family (of TLR1-13) and the nature of their ligands, have shed light on many aspects of pathogen recognition. It is clear that via differential expression of PRR, cells of the innate immune system of both mouse and humans have the ability to recognise pathogenic lipids and carbohydrates and remarkably PRR also enable the recognition of nucleic acids of both pathogenic and self origin.
The recognition of nucleic acids can be via 4 different TLR; TLR3 recognizes dsRNA, TLR7, and TLR 8 (truncated in the mouse) recognize ssRNA and TLR9 recognizes ssDNA. The TLR 7, 8, and 9-dependent recognition of nucleic acids occurs within cellular endosomes and is critically dependent upon the adaptor molecule MyD88. Non-TLR dependent nucleic acid recognition pathways also exist, but are less well defined than the TLR-mediated recognition pathways. RNA recognition that is TLR and MyD88-independent occurs via a cytoplasmic localised recognition complex involving RigI and Mda-5 molecules and can also involve the cytoplasmic Nod-like receptors. The cytoplasmic recognition of DNA, at least B-DNA (right-handed B-form DNA), is independent of the RigI and Mda-5 molecules, but shares with the Rig pathway downstream signalling molecules including TBK-1 and IKKi and can include molecules such as DAI and 3′ repair exonuclease 1 (Trex1). An inflammasome complex mediated by binding of AIM2 to dsDNA can also be involved in sensing of cytoplasmic dsDNA, leading to caspase-1 dependent cleavage of IL-1β.
The pDC of both mouse and humans recognise DNA via TLR9. As a consequence of endoplasmic reticulum to lysosome internal trafficking of TLR9 and differential expression of molecules that are involved in the TLR9 signalling complex, such as high constitutive expression of IRF7, the pDC, unlike any other cell previously described, have the ability to produce extremely high levels of IFN-I upon TLR9 ligation. Synthetic CpG-containing oligonucleotides (ODN) are sufficient for triggering of IFN-I from pDC and, in fact, the pDC are the only cell type known to produce IFN-I to CpG-ODN. This statement holds true in mouse in most lymphoid organs. However, as previously shown, when bone marrow (BM) cells are depleted of pDC, there remains IFN-α production in response to CpG-ODN (Hochrein et al. 2004). These data suggested that cells other than CD45R+CD45RA+ pDC were capable of TLR9-mediated IFN-α production. This finding contradicts the current dogma that pDC are the only cell type that produce IFN-I in response to TLR9 mediated ligation. The biological relevance of this observation extends beyond responses to CpG-ODN; many viruses have now been shown to activate cells via TLR9-mediated recognition (Hochrein and O'Keeffe 2008) and leaves the possibility that another cell type in BM could respond rapidly to TLR9 ligation with high levels of IFN-α production.
The BM is the birthplace of hematopoiesis and the source of life-long stem cells. It is also a haven for plasma cells and memory T cells and the cells involved in bone morphogenesis. However, it is also a site frequently infected by viruses, and yet the knowledge of the cellular responses to viruses or other pathogens in the bone marrow is extremely limited. With the advent of BM transplantation and a desire to understand the cellular entities potentially involved in transplantation rejection, it is of upmost importance to clarify the cell types within BM.
It has been reported that human early pre-pDC, that do not share the pDC phenotype, also have the ability to produce high levels of Type I IFN (Chen et al. 2004). These interferon producing cells (IPCs) exhibited a plasmacytoid morphology. The IPCs were also CD11c− and strongly expressed TLR9. The paper also reports the isolation of immature DC that were CD11c+ and preferentially expressed TLR3. IPC, but not CD11c+ immature DC, could produce high levels of IFN-α upon stimulation with Herpes simplex virus. The CD11c+ immature DC described in Chen et al. 2004 did not express TLR9 and produced little IFN-α in response to Herpes Simplex virus. Thus these cells cannot respond rapidly to TLR9 ligation with high levels of IFN-α production.
When spleen, thymus, mesenteric or subcutaneous lymph nodes are depleted of pDC using pDC-specific antibodies and magnetic beads, the IFN-α activity in response to CpG-ODN is abolished. However, when bone marrow (BM) cells are depleted of pDC there remains IFN-α production in response to CpG-ODN (Hochrein et al 2004).
Based on the above, there is a need in the art for the isolation of cell types other than pDC that can respond rapidly to TLR9 ligation with high levels of IFN-α production.